PROJECT SUMMARY Rapidly dividing cells must produce 2,000 new ribosomes every minute, and ensure that they are fully functional. Escape of incompletely assembled ribosomes into the translating pool has been suggested to underlie the high cancer incidence observed in patients suffering from Diamond Blackfan Anemia, 5q- syndrome and congenital asplenia. These considerations suggest that in healthy cells mechanisms must be in place to ensure that only correctly assembled ribosomes are released into the translating pool. In the previous funding period, we have discovered such a quality control mechanism, which uses the translational machinery to test the functionality of assembling 40S subunits: Pre-40S subunits are joined by mature 60S subunits in a reaction promoted by the translation factor eIF5B. Assembly factors dissociate from these 80S-like ribosomes, which do not produce protein, as they do not contain mRNA or tRNA. Release of assembling subunits into the translating pool is gated by the termination factors Rli1 and Dom34. However, exactly how progression of this cascade is linked to successful completion of functional tests is unknown, as is the mechanism by which assembly factors are released from 80S-like ribosomes. Here we address the hypothesis that assembly factors and translation factors cooperate to release assembly factors, and induce conformational changes in assembling ribosomes that are akin to those that underlie its function during translation. In Aim 1 we will dissect how the ATPase activity of the assembly factor Rio2 is used to trigger conformational changes in pre- 40S subunits that allow for joining of 60S subunits. In Aim 2 we will test how the translation factor eIF5B is used to release the assembly factor Rio2 from pre-40S subunits. In Aim 3 we will discern how the assembly factor Fap7 and the translation factor eEF2 cooperate to promote conformational changes used during translation for the translocation of tRNA and mRNA. These experiments take advantage findings and reagents produced during the last funding period, and use a unique combination of genetic, biochemical and structural experiments to address these fundamental questions.